Therapy for Transthyretin-Associated Amyloidosis

ABSTRACT

It is provided a catechol-O-methyltransferase (COMT) inhibitor for use in the prevention and/or treatment of transthyretin-associated amyloidosis. It is also provided a catechol-O-methyltransferase (COMT) inhibitor for use in the prevention and/or treatment of transthyretin-associated amyloidosis in combination therapy with another COMT inhibitor, a benzoxazole derivative, iododiflunisal, diflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline, or epigallocatechin-3-gallate.

The present invention is associated to the field of amyloid diseasesand, particularly, to new compounds for the prevention and/or treatmentof transthyretin-associated amyloidosis.

BACKGROUND ART

Amyloidosis refers to a variety of conditions in which amyloid proteinsare abnormally deposited in organs and/or tissues. These amyloidproteins sometimes exist in an abnormal fibre-like form, called amyloidfibrils or amyloid deposits, that build up and progressively interferewith the structure and function of affected organs throughout the body.Different proteins are implicated in different types of amyloid disease,and treatment depends on the particular amyloid protein.

Transthyretin-associated amyloidosis is a general denomination for agroup of amyloid diseases that are specifically associated totransthyretin abnormal misfolding, aggregation (fibril formation) andsubsequent deposition. Transthyretin (TTR) protein is a serum andcerebrospinal fluid carrier of the thyroid hormone thyroxine andretinol. Mutations in the TTR gene, which is located on human chromosome18q12.1-11.2, sometimes result in a destabilization of the TTR protein,leading to abnormal aggregation and transthyretin-associated amyloiddisease. More than 80 amyloid forming variants of TTR are known, ofwhich the most frequent is called TTR V30M.

Familial amyloid polyneuropathy (FAP), also calledtransthyretin-associated hereditary amyloidosis, transthyretinamyloidosis or Corino de Andrade's disease, is an autosomal dominantneurodegenerative disease. Usually manifesting itself between 20 and 40years of age, it is characterized by pain, paresthesia, muscularweakness and autonomic dysfunction. In its terminal state, the kidneysand the heart are affected. FAP is characterized by the systemicdeposition of amyloid variants of the TTR protein, especially in theperipheral nervous system, causing a progressive sensory and motorialpolyneuropathy. This disease is by far the most common type ofhereditary amyloidosis in the world.

Other types of transthyretin-associated amyloidosis are familial amyloidcardiomyopathy and senile systemic amyloidosis, caused by the depositionof amyloid TTR in the heart, and leptomeningeal amyloidosis, whereamyloid deposits of TTR are found in the walls of leptomeningealvessels, in pia-arachnoid, and also in subpial space deposits. Thelatter condition is associated with a clinical picture of centralnervous system impairment manifest as dementia, ataxia, and spasticity.

Whilst there is, as yet, no treatment that blocks amyloid deposition orspeeds up its removal, treatment of amyloid diseases is aimed atsupporting the function of failing organs. Liver transplantation hasbeen often used as a treatment for transthyretin-associated amyloidosis,particularly FAP, since TTR protein is mainly produced in the liver.Replacement of the liver containing a mutant TTR gene by a liver thatmakes normal transthyretin protein is aimed at preventing the formationof further amyloid and can stabilise the disease. Liver transplantationhas been performed in patients with FAP, with great success in manycases. However, a liver transplantation is not always an availableoption and, besides, as experience increases, it is becoming clear thatliver transplantation for FAP should take place before too much damageto the nerves or heart has already occurred. Sadly, the latter may occurwithout causing any symptoms.

Very few compounds have been described as exerting an inhibitoryactivity against fibril formation and subsequent deposition of TTR.Among these, iododiflunisal has been reported as a potent amyloidinhibitor in vitro by Gales et al (Gales L, Macedo-Ribeiro S, ArsequellG, Valencia G, Saraiva M J, Damas A M. “Human transthyretin in complexwith iododiflunisal: structural features associated with a potentamyloid inhibitor”. Biochem J, 2005, vol. 388, p. 615-621). Further,patent application WO 2005/113523 discloses benzoxazole compounds forstabilizing TTR amyloid protein, thus preventing the formation of TTRamyloid fibrils. These compounds are claimed as useful for the treatmentof transthyretin-associated amyloid diseases.

Particularly, a benzoxazole derivative called tafamidis(2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid has beendescribed to inhibit TTR abnormal aggregation and fibril formation, andis undergoing clinical trials for the treatment of FAP. Despite being apromising perspective, tafamidis is still under evaluation in the mainofficial medicine registration offices. Thus, the clinical relevance oftafamidis still needs to be clarified.

Despite the considerable effort that has been made in the field, thereis to date no effective pharmacological therapy for the treatment ofFAP. It is therefore desirable to provide alternative compounds for thetreatment of FAP and other transthyretin-associated amyloidosis.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that catechol-O-methyltransferase(COMT) inhibitors are useful for the prevention and/or treatment ofTTR-associated amyloidosis.

As shown in the examples below, the COMT inhibitor tolcapone has a highinhibiting activity against TTR amyloid formation. The good inhibitoryactivity of tolcapone is revealed by its low IC₅₀ and high percentamyloidosis reduction (RA %) values.

Thus, a first aspect of the present invention relates to a COMTinhibitor for use in the prevention and/or treatment of TTR-associatedamyloidosis. This aspect can be reformulated as use of a COMT inhibitorfor the preparation of a medicament for the prevention and/or treatmentof a TTR-associated amyloidosis.

It also forms part of the invention a method for the prevention and/ortreatment of a TTR-associated amyloidosis comprising administering aCOMT inhibitor to a subject in need thereof. In a particular embodiment,the subject in need of the prevention and/or treatment is a mammal,including a human. In a further preferred embodiment, the mammal is ahuman.

As compared to tafamidis, which is so far the most advancedpharmacological compound for FAP treatment, tolcapone has a four foldlower IC₅₀ in vitro, which means that the concentration of tolcaponeneeded to inhibit 50% of TTR fibril formation is much lower than that oftafamidis (see examples below). The examples below additionallydemonstrate that tolcapone binds to TTR and prevents TTR-inducedcytotoxicity to a greater extent than tafamidis.

According to these results, tolcapone is more effective in reducing TTRfibril formation than the reference tafamidis compound. In addition topreventing TTR fibril formation, the inventors have found that tolcaponeexhibits an important disruption activity over existing TTR fibrils. Theresults presented below demonstrate that tolcapone's TTR fibrildisruption activity is higher than that of tafamidi's.

COMT inhibitors are well known in the state of the art as compounds thatinhibit the action of catechol-O-methyl transferase, an enzyme that isinvolved in degrading neurotransmitters (Mannisto and Kaakkola, Pharm.Rev., 1999, vol 51, p. 593-628). COMT inhibitor activity can bedetermined by methods known in the art, for instance the methoddisclosed in Zürcher et al (Biomedical Chromatography, 1996, vol. 10, p.32-36). COMT inhibitors are well known in the art of pharmacology forthe treatment of Parkinson's disease in conjunction with dopaminergicagents such as L-DOPA.

Several COMT inhibitors have been described. Tolcapone, entacapone, andnitecapone belong to the so called “second generation COMT inhibitors”,which have been shown to be potent, highly selective, and orally activeCOMT inhibitors. Nitrocatechol is the key structure in these molecules(Pharm. Rev., 1999, vol 51, p. 593-628, supra). Thus, in one embodimentthe COMT inhibitor for use in the prevention and/or treatment ofTTR-associated amyloidosis is a nitrocatechol compound. In a particularembodiment, the nitrocatecol compound has the following formula I

or a pharmaceutically acceptable salt thereof, wherein R=—C(O)-PhCH₃,—CH═C(N)—C(O)—NEt₂ or —CH═C(O)(CH₃)—C(O)(CH₃).

In another embodiment of the first aspect of the invention the COMTinhibitor is tolcapone, entacapone or nitecapone, or pharmaceuticallyacceptable salts thereof.

In a particular embodiment the COMT inhibitor is tolcapone, or apharmaceutically acceptable salt thereof. Tolcapone (formula II) is ayellow, odorless, non-hygroscopic, crystalline compound with a relativemolecular mass of 273.25. Its empirical formula is C₁₄H₁₁NO₅. Thechemical name of tolcapone is3,4-dihydroxy-4′-methyl-5-nitrobenzophenone and its CAS reference numberis 134308-13-7.

In another embodiment of the first aspect of the invention the COMTinhibitor is entacapone, or a pharmaceutically acceptable salt thereof.Entacapone (formula III) is a yellow crystalline compound with molecularmass of 305.29. Its empirical formula is C₁₄H₁₅N₃O₅. The chemical nameof entacapone is(2E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-2-propenamideand its CAS reference number is 130929-57-6.

Since these compounds are drugs that have been approved for medical usein the treatment of Parkinson Disease by the Food and DrugAdministration (FDA) and European Medicines Agency (EMA) since 1998, thebioavailability and safety profile of tolcapone and entacapone have beenstudied in several clinical trials. As such, these compounds have anacceptable safety profile for human use and good bioavailability. Theirsafety profile in conjunction with their high inhibitory activityagainst TTR fibril formation render the COMT inhibitors highly promisingdrugs for the prevention and/or treatment of TTR-associated amyloidosis.

Additionally, since these compounds have already been subjected toclinical trials for the treatment of human disease, the clinicalpoof-of-concept is less risky (and faster) to achieve when compared withclassical development of new chemical entities. In this sense, it isimportant to highlight that considerable fewer experimentation needs tobe done in human beings and animals, subsequently implying lowerdevelopmental costs and, more importantly, less sufferings to humans andanimals.

In another embodiment of the first aspect of the invention the COMTinhibitor is nitecapone, or a pharmaceutically acceptable salt thereof.Nitecapone (formula IV) is a compound with molecular mass of 265.21. Itsempirical formula is C₁₂H₁₁NO₆, the chemical name3-[(3,4-Dihydroxy-5-nitrophenyl)methylene]-2,4-pentanedione, and CASreference number 116313-94-1.

In a preferred embodiment of the invention the TTR-associatedamyloidosis is FAP. In another embodiment the TTR-associated amyloidosisis senile systemic amyloidosis. In another embodiment the TTR-associatedamyloidosis is familial amyloid cardiomyopathy. In yet anotherembodiment the TTR-associated amyloidosis is leptomeningeal amyloidosis.

COMT inhibitors, such as those defined above can be used either alone orin combination with other therapeutic agents for the prevention and/ortreatment of TTR-associated amyloidosis. Thus, in a second aspect, theinvention refers to a combination of a COMT inhibitor and an additionaltherapeutic agent for the prevention and/or treatment of aTTR-associated amyloidosis. This embodiment can be reformulated as acombination of a COMT inhibitor and an additional therapeutic agent forthe prevention and/or treatment of a TTR-associated amyloidosis.Further, it also forms part of the invention a method for the preventionand/or treatment of a transthyretin-associated amyloidosis whichcomprises administering to a subject in need thereof a combination of aCOMT inhibitor and an additional therapeutic agent. Non-limitingexamples of additional therapeutic agents for use in the second aspectof the invention are another COMT inhibitor, a benzoxazole derivative,iododiflunisal, diflunisal, resveratrol, tauroursodeoxycholic acid,doxocycline and epigallocatechin-3-gallate (EGCG). Preferably, the COMTinhibitor is a nitrocatechol compound of formula I or a pharmaceuticallyacceptable salt thereof as defined for the first aspect of theinvention. The skilled person will understand that pharmaceuticallyacceptable salts of the above mentioned additional therapeutic agentscan also be used in the combination of the second aspect of theinvention.

In one embodiment of the second aspect of the invention it is provided acombination of a COMT inhibitor and an additional therapeutic agentselected from the group consisting of another COMT inhibitor, abenzoxazole derivative, and iododiflunisal for use in the preventionand/or treatment of transthyretin-associated amyloidosis. Preferably,the COMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention.

Benzoxazole derivatives are disclosed in the international patentapplication WO2005113523 as compounds that stabilize the native state ofTTR, thereby inhibiting protein misfolding. In one embodiment of thesecond aspect of the invention, the benzoxazole derivatives arecompounds of formula V:

or a pharmaceutically acceptable salt thereof, wherein:Y is COOR, tetrazolyl, CONHOR, B(OH)₂ or OR;

X is O; and

R¹, R² and R³ are each independently selected from hydrogen, halo, OR,B(OH)₂ or CF₃, andwherein R is hydrogen, C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆cycloalkyl, C₁-C₆ heterocyclyl, phenyl, xylyl, naphthyl, thienyl,indolyl or pyridyl.

In a particular embodiment of the second aspect of the invention theCOMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention and the additional therapeutic agent is a benzoxazolederivative of formula V or a pharmaceutically acceptable salt thereof asdefined above.

In another embodiment of the second aspect of the invention thebenzoxazole derivative is a compound of formula VI

or a pharmaceutically acceptable salt thereof, wherein:

Y is COOH, or OH; and

R¹, R² and R³ are each independently selected from hydrogen, halo, OH,B(OH)₂ or CF₃.

In a particular embodiment the COMT inhibitor is a nitrocatecholcompound of formula I or a pharmaceutically acceptable salt thereof asdefined for the first aspect of the invention and the additionaltherapeutic agent is a benzoxazole derivative of formula VI or apharmaceutically acceptable salt thereof as defined above.

In another embodiment the benzoxazole derivative is tafamidis. In aparticular embodiment the COMT inhibitor is a nitrocatechol compound offormula I or a pharmaceutically acceptable salt thereof as defined forthe first aspect of the invention and the additional therapeutic agentis tafamidis. In another particular embodiment the COMT inhibitor istolcapone or a pharmaceutically acceptable salt thereof and theadditional therapeutic agent is tafamidis.

In another embodiment of the second aspect of the invention, theadditional therapeutic agent is iododiflunisal. In a particularembodiment the COMT inhibitor is a nitrocatechol compound of formula Ior a pharmaceutically acceptable salt thereof as defined for the firstaspect of the invention and the additional therapeutic agent isiododiflunisal. In another particular embodiment the COMT inhibitor istolcapone or a pharmaceutically acceptable salt thereof and theadditional therapeutic agent is iododiflunisal.

In another particular embodiment, the COMT inhibitor is combined withanother COMT inhibitor. Preferably, the COMT inhibitors arenitrocatechol compounds of formula I or pharmaceutically acceptablesalts thereof as defined for the first aspect of the invention. Forinstance, the invention provides a combination of tolcapone andentacapone for the prevention and/or treatment of a TTR-associatedamyloidosis.

In a further embodiment of the second aspect of the invention it isprovided a combination of a COMT inhibitor and an additional therapeuticagent selected from the group consisting of diflunisal, resveratrol,tauroursodeoxycholic acid, doxocycline and EGCG for use in theprevention and/or treatment of a TTR-associated amyloidosis. EGCG is athe main and most significant polyphenol in green tea. In the sense ofthe present invention, EGCG can be used as an isolated compound orforming part of a plant extract, particularly a tea extract. Preferably,the COMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention. More preferably the COMT inhibitor is tolcapone or apharmaceutically acceptable salt thereof. A particular embodimentprovides a combination of tolcapone or a pharmaceutically acceptablesalt thereof and EGCG for use in the prevention and/or treatment of aTTR-associated amyloidosis.

As will be apparent to the skilled in the art, the combination of thepresent invention is effective not only when the active ingredients areused in a single composition, but also when used in two differentcompositions, either administered simultaneously, sequentially orseparately after a certain period of time. Furthermore, the skilled inthe art will understand that the COMT inhibitor can be prescribed to beused together with the other active ingredient in a combination therapyin order to prevent and/or treat a transthyretin-associated amyloidosis,and vice versa.

Thus, a third aspect of the present invention provides a COMT inhibitorfor use in the prevention and/or treatment of transthyretin-associatedamyloidosis in combination therapy with an additional therapeutic agent.This embodiment may be reformulated as use of a COMT inhibitor for thepreparation of a medicament for the prevention and/or treatment oftransthyretin-associated amyloidosis in combination therapy with anadditional therapeutic agent. It also forms part of the invention amethod for the prevention and/or treatment of a transthyretin-associatedamyloidosis which comprises administering to a subject in need thereof aCOMT inhibitor in combination with an additional therapeutic agent.

Non-limiting examples of additional therapeutic agents for use in thethird aspect of the invention are another COMT inhibitor, a benzoxazolederivative, iododiflunisal, diflunisal, resveratrol,tauroursodeoxycholic acid, doxocycline and EGCG. Preferably, the COMTinhibitor is a nitrocatechol compound of formula I or a pharmaceuticallyacceptable salt thereof as defined for the first aspect of theinvention. The skilled person will understand that pharmaceuticallyacceptable salts of the above mentioned additional therapeutic agentscan also be used in the combination therapy of the third aspect of theinvention.

In one embodiment of the third aspect of the invention it is provided aCOMT inhibitor for use in the prevention and/or treatment oftransthyretin-associated amyloidosis in combination therapy with anadditional therapeutic agent selected from the group consisting ofanother COMT inhibitor, a benzoxazole derivative, and iododiflunisal.Preferably, the COMT inhibitor is a nitrocatechol compound of formula Ior a pharmaceutically acceptable salt thereof as defined for the firstaspect of the invention.

In a particular embodiment of the third aspect of the invention, theadditional therapeutic agent is another COMT inhibitor. Preferably, theCOMT inhibitors are nitrocatechol compounds of formula I orpharmaceutically acceptable salts thereof as defined for the firstaspect of the invention. For example, the invention provides tolcaponefor the prevention and/or treatment of a TTR-associated amyloidosis incombination with entacapone. In another particular embodiment, theadditional therapeutic agent is a benzoxazole derivative.

Preferably, said benzoxazole derivative is a compound of formula V or VIor pharmaceutical salts thereof as defined for the second aspect of theinvention. For example, the invention provides a COMT inhibitor for theprevention and/or treatment of a TTR-associated amyloidosis incombination with tafamidis. Preferably, the COMT inhibitor is anitrocatechol compound of formula I or a pharmaceutically acceptablesalt thereof as defined for the first aspect of the invention. Morepreferably the COMT inhibitor is tolcapone or a pharmaceuticallyacceptable salt thereof. Thus the invention provides tolcapone for theprevention and/or treatment of a TTR-associated amyloidosis incombination with tafamidis. In yet another embodiment, the additionaltherapeutic agent is iododiflunisal. In yet another embodiment, theadditional therapeutic agent is iododiflunisal and the COMT inhibitor isa nitrocatechol compound of formula I or a pharmaceutically acceptablesalt thereof as defined for the first aspect of the invention. Theinvention thus provides tolcapone or a pharmaceutically acceptable saltthereof for the prevention and/or treatment of a TTR-associatedamyloidosis in combination with iododiflunisal.

In a further embodiment of the third aspect of the invention it isprovided a COMT inhibitor for use in the prevention and/or treatment oftransthyretin-associated amyloidosis in combination therapy with anadditional therapeutic agent selected from the group consisting ofdiflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline and EGCGfor use in the prevention and/or treatment of a TTR-associatedamyloidosis. Preferably, the COMT inhibitor is a nitrocatechol compoundof formula I or a pharmaceutically acceptable salt thereof as definedfor the first aspect of the invention. More preferably the COMTinhibitor is tolcapone or a pharmaceutically acceptable salt thereof. Ina particular embodiment the invention provides tolcapone or apharmaceutically acceptable salt thereof for use in the preventionand/or treatment of transthyretin-associated amyloidosis in combinationtherapy with EGCG.

A fourth aspect of the invention provides a therapeutic agent selectedfrom the group consisting of a benzoxazole derivative, iododiflunisal,diflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline andEGCG, for use in the prevention and/or treatment oftransthyretin-associated amyloidosis in combination therapy with a COMTinhibitor. Preferably, the COMT inhibitor is a nitrocatechol compound offormula I or a pharmaceutically acceptable salt thereof as defined forthe first aspect of the invention. The skilled person will understandthat pharmaceutically acceptable salts of the above mentionedtherapeutic agents can also be used in the combination therapy of thefourth aspect of the invention.

In one embodiment of the fourth aspect of the invention it is provided atherapeutic agent selected from the group consisting of a benzoxazolederivative and iododiflunisal for use in the prevention and/or treatmentof transthyretin-associated amyloidosis in combination therapy with aCOMT inhibitor. Preferably, the COMT inhibitor is a nitrocatecholcompound of formula I or a pharmaceutically acceptable salt thereof asdefined for the first aspect of the invention.

In a particular embodiment of the fourth aspect of the invention, thetherapeutic agent is a benzoxazole derivative. Preferably, saidbenzoxazole derivative is a compound of formula V or VI orpharmaceutical salts thereof as defined for the second aspect of theinvention. For example, the invention provides tafamidis for theprevention and/or treatment of TTR-associated amyloidosis in combinationwith a COMT inhibitor. Preferably, the COMT inhibitor is a nitrocatecholcompound of formula I or a pharmaceutically acceptable salt thereof asdefined for the first aspect of the invention. More preferably the COMTinhibitor is tolcapone or a pharmaceutically acceptable salt thereof.Thus the invention provides tafamidis for the prevention and/ortreatment of a TTR-associated amyloidosis in combination with tolcaponeor a pharmaceutically acceptable salt thereof. In yet anotherembodiment, the therapeutic agent is iododiflunisal. In yet anotherembodiment, the additional therapeutic agent is iododiflunisal and theCOMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention. The invention provides iododiflunisal for theprevention and/or treatment of a TTR-associated amyloidosis incombination with tolcapone or a pharmaceutically acceptable saltthereof.

In a further embodiment of the fourth aspect of the invention it isprovided a therapeutic agent selected from the group consisting ofdiflunisal, resveratrol, tauroursodeoxycholic acid, doxocycline and EGCGfor use in the prevention and/or treatment of transthyretin-associatedamyloidosis in combination therapy with a COMT inhibitor. Preferably,the COMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention. More preferably, the COMT inhibitor is tolcapone or apharmaceutically acceptable salt thereof. A particular embodimentprovides EGCG for use in the prevention and/or treatment of aTTR-associated amyloidosis in combination therapy with tolcapone or apharmaceutically acceptable salt thereof.

Additionally, the COMT inhibitor can be used as adjuvant treatmentbefore and/or after liver transplant in a patient with a TTR-associatedamyloidosis. Preferably, said COMT inhibitor is a nitrocatechol compoundof formula I or a pharmaceutically acceptable salt thereof as definedfor the first aspect of the invention.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of a COMT inhibitor together withpharmaceutically acceptable excipients and/or carriers for theprevention and/or treatment of a TTR-associated amyloidosis. Preferably,said COMT inhibitor is a nitrocatechol compound of formula I or apharmaceutically acceptable salt thereof as defined for the first aspectof the invention.

The expression “therapeutically effective amount”, also referred as“dose”, refers to the amount of a compound that, when administered, issufficient to prevent development of, or alleviate to some extent, oneor more of the symptoms of the disease which is addressed. Theparticular dose of compound administered according to this inventionwill be determined by the particular circumstances surrounding the case,including the compound administered, the route of administration, theparticular condition being treated, and similar considerations.

The expression “pharmaceutically acceptable excipients and/or carriers”refers to pharmaceutically acceptable materials, compositions orvehicles. Each component must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of thepharmaceutical composition. It must also be suitable for use in contactwith the tissue or organ of humans and animals without excessivetoxicity, irritation, allergic response, immunogenicity or otherproblems or complications commensurate with a reasonable benefit/riskratio.

Any pharmaceutically acceptable salt of the COMT inhibitor can be usedfor the purposes of the invention. The term “pharmaceutically acceptablesalt” refers to salts prepared from pharmaceutically acceptablenon-toxic bases. Preferably, the salt is an alkaline or alkaline earthmetal salt.

In one embodiment of the invention, the COMT inhibitor is administeredto a patient in oral unit dosage form. Dosage forms include solid dosageforms like tablets, powders, capsules, sachets, as well as liquidsyrups, suspensions and elixirs. COMT inhibitors and excipients can beformulated into compositions and dosage forms according to methods knownin the art. In a particular embodiment, the COMT inhibitor isadministered as a tablet, a pill or a capsule. However, COMT inhibitorscan also be administered to a patient as an ingredient of injectiondosage forms. Injection dosage forms can include liquids forintradermal, intravenous, intramuscular or subcutaneous injection,solutions for perfusion, powder for reconstitution of liquid injections,and pre-filled syringes. In the sense of the present invention it mayalso be adequate to formulate the COMT inhibitor for intranasal orinhaled administration, or for topic administration in the form of, forinstance, a cream, a gel, an ointment or a dermal patch. Methods for thepreparation of these formulations are known in the art. Further, theCOMT inhibitor can be formulated as a controlled release dosage form.Controlled release dosage forms are known in the art and particularlydesirable for the treatment of chronic diseases or for theadministration of active agents that can be toxic at high doses or thatshow a low half-life pattern when administered to the patient.Preferably, the COMT inhibitor is a nitrocatechol compound of formula Ior a pharmaceutically acceptable salt thereof as defined for the firstaspect of the invention.

As mentioned above, a therapeutically effective amount (or dose) of COMTinhibitor in the sense of the present invention is the amount of saidcompound that is sufficient to prevent or alleviate to some extent oneor more of the symptoms of a TTR-associated amyloidosis. For instance,an effective daily dose of tolcapone for human use could range between20 and 600 mg and an effective daily dose of entacapone for human usecould range between 1600 and 2000 mg.

Thus, the dose of COMT inhibitor to be administered can be between 0.1and 16000 mg/day, or between 0.1 and 12000 mg/day, or between 0.1 and10000 mg/day, or between 0.1 and 5000 mg/day, or between 0.1 and 3000mg/day. In a particular embodiment, the dose of COMT inhibitor to beadministered is between 1 and 3000 mg/day. In another embodiment, thedose is between 1 and 2000 mg/day. Preferably, the COMT inhibitor is anitrocatechol compound of formula I or a pharmaceutically acceptablesalt thereof as defined for the first aspect of the invention.

Throughout the description and claims the word “comprise” and variationsof the word, are not intended to exclude other technical features,additives, components, or steps. Additional objects, advantages andfeatures of the invention will become apparent to those skilled in theart upon examination of the description or may be learned by practice ofthe invention. The following examples are provided by way ofillustration, and they are not intended to be limiting of the presentinvention. Furthermore, the present invention covers all possiblecombinations of particular and preferred embodiments described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Assay of competition with T4 for the binding to TTR wild type(WT) by gel filtration: Curves of T4 displacement from TTR WT bydifferent compounds. Y axis: Amount of TTR-bound T4/total T4; X-axis:log 10 concentration of compound (molar units). Values correspond to arepresentative experiment done in duplicates, represented asaverage+/−standard deviation. Test compounds: Thyroxine (T4), Tolcapone(SOM), Tafamidis (TAF), and (−)-epigallocatechin-3-gallate (EGCG).

FIG. 2. TTR tetrameric stability in the presence of different compoundsby IEF: Plasma from control individuals (C) and from familial amyloidpolyneuropathy patients carrying V30M mutation (V30M) was treated withtest compounds Tafamidis (T); tolcapone (S); epigallocatechin-3-gallate(EGCG) or left untreated (nt); and subjected to IEF under semidenaturingconditions as described in the text. The ratio of TTR tetramer/total TTRfor each condition was calculated and represented as average+/−sem(standard error of the mean).

FIG. 3: Caspase-3 activation. Rat Schwannoma cells (RN22 cell line) wereincubated 24 h in the absence or presence of TTR Y78F oligomers obtainedin the absence or presence of tested compounds (at 20 μM). Activation ofCaspase-3 was measured in cell lysates, and expressed asfluorescence/protein content. Samples: control cells (C1); Cells treatedwith EGCG (C2); Cells treated with tafamidis (C3); cells treated withtolcapone (C4); control cells treated with oligomer obtained in theabsence of compounds (O1); cells treated with oligomer obtained in thepresence of EGCG (O2); cells treated with oligomer obtained in thepresence of tafamidis (O3); cells treated with oligomer obtained in thepresence of tolcapone (O4). Results represent average of 4 replicatesand standard deviation. Significant differences respect O1 control werecalculated with T-student test: *: P<0.05; ***: P<0.005.

FIG. 4: Transmission Electron Microscopy analysis of preformed TTRfibrils after 4 days incubation with different compounds at 36 μM. Fromup left, clockwise: control, tafamidis, EGCG, Tolcapone.

EXAMPLES Example 1: Kinetic Turbidity Assay Materials

Recombinant Y78F TTR protein, which is a Tyr78Phe highly amyloidogenicvariation of human TTR, was produced as reported in Dolado et al (DoladoI, Nieto J, Saraiva M J, Arsequell G, Valencia G, Planas A. “KineticAssay for High-Throughput Screening of In Vitro Transthyretin AmyloidFibrillogenesis Inhibitors”. J. Comb. Chem., 2005, vol. 7, p. 246-252).

Tolcapone was obtained from Santa Cruz Biotechnology, Inc.Iododiflunisal, was prepared from diflusinal (Sigma) by reaction withbis(pyridine)iodonium tetrafluoroborate (IPy₂BF₄) as described byBarluenga et al (Barluenga J, González J M, García-Martin M A, Campos PJ, Asensio G. “An expeditious and general aromatic iodination procedure.J Chem Soc Chem Commun, 1992, vol. 14, p. 1016-1017). Tafamidis can beprepared by the methods disclosed in the international patentapplication WO2005113523. Stocks of compounds assayed as inhibitors weredissolved in DMSO (spectrophotometry grade from Sigma) at 1.5 mMconcentration. Working solutions were prepared by diluting the stocksolution 1:4 in H2O/DMSO (2:1). In all cases, DMSO concentration wasadjusted to 5% (v/v) in the final reaction assay mixture.

Methods

The assay was performed according to the procedure described in Doladoet al (supra). The assay comprises two stages, one stage where the Y78Fprotein is incubated together with the inhibitor during 30 minutes, anda second stage where fibril formation is induced by a change in pH andabsorbance is measured along 1.5 h. Briefly, the assay was performed asfollows:

First, the following solutions were prepared: Protein Y78F stock: 4mg/mL in 20 mM phosphate, 100 mM KCl, pH 7.6. Incubation buffer: 10 mMphosphate, 100 mM KCl, 1 mM EDTA, pH 7.6. Dilution buffer: 400 mM sodiumacetate, 100 mM KCl, 1 mM EDTA, pH 4.2.

For each inhibitor the following protocol was followed: Exact proteinconcentration of the stock solution was determined by Abs₂₈₀ andaccording to this value, the volume of Y78F stock to be added to have afinal protein well concentration of 0.4 mg/mL was calculated anddispensed into 6 wells of a 96-well microplate. Different volumes ofworking inhibitor solution were added to give final concentrationsranging from 0 to 40 μM, and the final DMSO content of each well wasadjusted to 5% by adding the corresponding volume of a H₂O/DMSO (1:1)solution. Incubation buffer was then added up to a volume of 100 μL. Theplate was incubated at 37° C. in a thermostated microplate reader withorbital shaking 15 s every minute for 30 min. A 100 μL portion ofdilution buffer was dispensed to each well, and the mixture wasincubated at 37° C. with shaking (15 s every min) in the microplatereader. Absorbance at 340 nm was monitored for 1.5 h at 1 min intervals.Data were collected and analyzed using Microsoft Excel software. Allassays were done in duplicate.

Result Analysis

After following the general procedure indicated above, time-coursecurves were obtained, from which the initial rates of fibril formation(V₀) were calculated as the slopes of the linear increase of absorbance.When plotting the initial rates vs inhibition concentration, anexponential decay was obtained with all inhibitors analyzed. Data werefitted to equation (1):

V ₀ =A+B*e ^(−C[I])  (1),

where V₀ is the initial rate of fibril formation (in absorbance unitsper hour, Abs*h⁻¹), and [I] is the concentration of the inhibitor (μM).Adjustable parameters are A (Abs*h⁻¹), residual aggregation rate at highconcentration of inhibitor; B (Abs*h⁻¹), amplitude or maximum decreaseof initial rate of fibril formation; and C (μM⁻¹), the exponentialconstant. A+B is equal to the initial rate of fibril formation under theassay conditions in the absence of inhibitor.

The following parameters were estimated to evaluate the potency of acompound as fibril formation inhibitor: IC₅₀: concentration of inhibitorat which the initial rate of fibril formation is one-half that withoutinhibitor. RA (%)=100*B/(A+B): percent reduction of fibril formationrate at high inhibitor concentration relative to the rate at [I]=0.Results of evaluation of the inhibition properties of assayed compoundsare summarized in Table 1.

TABLE 1 IC₅₀ and percentage of amyloidosis reduction (RA) values for TTRfibril formation inhibitors Compound IC₅₀ (μM) RA (%) Tolcapone 4.8 85.8Iododiflunisal 3.9 99.8 Tafamidis 16.9 99

It can be observed by the above results that tolcapone is an effectiveinhibitor of TTR fibril formation, as it showed a low IC₅₀ and a highRA. According to their IC₅₀ values, tolcapone has a similar inhibitioncapacity as compared with iododiflunisal, which has been reported as oneof the most potent TTR fibril formation inhibitors in vitro. Further,according to the IC₅₀, tolcapone is more effective than tafamidis, sinceit shows an IC₅₀ which is four times lower than tafamidis. These resultsdemonstrate that tolcapone is a promising drug for TTR-relatedamyloidosis, such as FAP, familial amyloid cardiomyopathy senilesystemic amyloidosis and leptomeningeal amyloidosis.

Example 2: End-Point Turbidity Assay with a Familiar AmyloidCardiomyopathy Mutant Variant of TTR Materials

Recombinant V122I TTR protein, which is an amyloidogenic variation ofhuman TTR associated with Familial Amyloid Cardiomyopathy (FAC), wasproduced by following the same procedure described for the Y78F variantused in Example 1. Plasmid DNA expressing the V122I mutant was preparedby site-directed mutagenesis as reported for Y78F in Dolado et al(supra). but using the following primers: 5′-GGATTGGTGATGACAGCCGT-3′ and5′-ACGGCTGTCATCACCAATCC-3′. Tolcapone and Iododiflunisal were obtainedas described in Example 1.

Methods

This assay is used for TTR variants with lower amyloidegenicity than theY78F variant when the kinetic turbidity assay is not sensitive enoughfor accurate measurements. The procedure followed to test the inhibitorsby this end-point assay at 72 h is reported in Dolado et al, (supra).V122I TTR was incubated with the inhibitor under the same conditionsdescribed above for the kinetic turbidity assay (Example 1), using V122Iprotein at a concentration of 0.4 mg/mL and three differentconcentrations of inhibitor: 3.6, 7.2 and 21.8 microM, corresponding to0.5×[protein], 1×[protein], and 3×[protein]. After acid induction(addition of dilution buffer), samples were incubated without shakingfor 72 h at 37° C. and then homogenized by mixing to resuspend anyfibrils present. Turbidity was measured at 340 nm and normalized toamyloidogenesis in the absence of inhibitor.

Result

The inhibitory potency of the tested compounds was evaluated as thepercentage of absorbance reduction of the inhibitor-containing sampleswhen compared with the inhibitor-free control sample.

TABLE 2 % Fibril Reduction values for V122I TTR fibril formationinhibitors Inhibitor concentration: 0.5x[protein] 1x[protein]3x[protein] Tolcapone 79.3% 84.3% 100.0% Iododiflunisal 83.2% 85.0%88.2% % Fibril reductions = 100 × (1 − turbidity sample/turbidityblank), where turbidity sample is the turbidity measured in the presenceof inhibitor, and turbidity blank is that in the absence of inhibitor.

The above results show that tolcapone effectively inhibits fibrilformation by V122I mutant ATTR, even at a inhibitor:protein molar ratioof 1:2 (0.5×[protein]). According to these values, tolcapone has asimilar inhibition capacity as compared with iododiflunisal. Theseresults demonstrate that tolcapone is a promising drug for TTR-relatedamyloidosis, including familial amyloid cardiomyopathy, which is causedmainly by the V122I mutation.

Examples 3-6 Materials for Examples 3-6

Tolcapone and tafamidis were obtained as described in example 1. TheEpigallocatechin-3-gallate (EGCG, CAS No. 989-51-5) was purchased fromCayman Chemicals (#70935). Recombinant wild-type TTR (TTR WT), TTR Y78Fand TTR L55P variants were produced in a bacterial expression systemusing Escherichia coli BL21. Recombinant TTRs were isolated and purifiedas previously described (Ferreira et al, 2009, FEBS Lett, vol. 583, p.3569-76). Whole blood from TTR V30M heterozygote carriers and fromcontrol individuals were obtained from a collection of samples availableat the Molecular Neurobiology Group, IBMC (University of Porto). Bloodsamples had been collected in the presence of EDTA and centrifuged forthe separation of plasma. Plasmas had been kept frozen at −20° C.

Example 3: Assay of Competition with Thyroxine (T4) for the Binding toTTR Wild Type (WT) by Gel Filtration

Binding of small molecule ligands to the T4 binding sites of TTR mightstabilize the TTR tetramer and slow tetramer dissociation andamyloidogenesis in vitro. To assess binding, competition of testcompounds with T4 (Sigma-Aldrich) for binding to TTR WT was assayedquantitatively by a gel filtration procedure, using a constant amount ofTTR (100 μL of 60 nM solution) incubated with a trace amount ofradiolabeled [125I]T4 (corresponding to 50.000 cpm; 125I-T4 specificactivity 1250 μCi/μg from Perkin-Elmer, MA, USA) and with 100 μL ofsolution of either test compounds or T4 (positive control) at differentconcentrations, namely 0, 20, 60, 200, 600, 2000 6000 and 20000 nM (0-10μM final concentration) (Ferreira et al, 2011, FEBS Lett., vol. 585, p.2424-30). The negative control was prepared with the protein, pluslabelled T4 plus 100 μL of THE (absence of competitor). All solutionswere prepared in THE buffer (Tris 0.1 M, NaCl 0.1 M, EDTA 1 mM). Allsamples were prepared in duplicate. Radioactivity was measured in eachsample, in a gamma scintillation counter Wizard 14701, Wallac. Thesamples were then incubated overnight at 4° C. After incubation, T4bound to TTR was separated from unbound T4 by filtration through a P6DGgel filtration column (1 mL, BioRad). Radioactivity was measured in theeluted samples. The results were expressed as the amount of TTR-boundT4/total T4 against Log total concentration of test compounds(competitors). Data was fitted to a one-site binding competitionnon-linear regression curve with GraphPad Prism software using thefollowing equation: Y=Bottom+(Top−Bottom)/(1+10̂(X−Log EC50))

FIG. 1 shows the results for competition with T4 for the binding to TTRwild type of competitors: Thyroxine (T4), Tolcapone (SOM), Tafamidis(TAF), and (−)-epigallocatechin-3-gallate (EGCG). The results are shownas the curves of T4 displacement from TTR WT by the different compounds.From each dose-response curve, the EC₅₀ value (inhibitor concentrationat which half of the bound T4 is displaced) for each compound isdetermined. Further, the relative potency for the inhibition of bindingof T4, defined as the ratio EC₅₀ (T4)/EC50 (tested compound), was alsocalculated and is shown in table 3.

TABLE 3 EC₅₀ and relative potency of drug inhibition of T4 bindingRelative potency of drug EC50 nM inhibition of T4 binding Thyroxine (T4)50.11 nM 1 Tolcapone 41.85 nM 1.19 Tafamidis 214.4 nM 0.23 EGCG — Noaffinity

These results demonstrate that tolcapone and tafamidis present similarbinding affinity to TTR, while EGCG does not compete with T4 for thebinding to TTR. The EC₅₀ of tolcapone was 4 times lower than that oftafamidis, which demonstrates that tolcapone is more effective inbinding the TTR tetramer, suggesting a higher anti-amyloidogenicpotential.

Example 4: Assessment of TTR Tetrameric Stability by IsoelectricFocusing (IEF)

To evaluate the effect of the tested compounds on TTR tetramerresistance to dissociation, TTR stability was assessed by IEF insemi-denaturing conditions as previously described (Ferreira et al,2009, FEBS Lett, vol. 583, p. 3569-76). Samples were prepared asfollows: 30 μL of human plasma from controls and TTR V30M carriers wereincubated with 5 μl of 10 mM solution of test compounds and control(EGCG) compounds overnight at 4° C. followed by a 1 h incubation at RT.The preparations were subjected to native PAGE (5% acrylamide) and thegel band containing TTR was excised and applied to an IEF gel (5%acrylamide). IEF was carried out in semi-denaturing conditions (4 Murea), containing 5% (v/v) ampholytes pH 4-6.5 (GE Healthcare), at 1200V for 6 hours. Proteins were stained with Coomassie Blue, the gels werescanned and subjected to densitometry using the ImageQuant program (HPScanjet 4470c, Hewlett Packard). In the absence of any compound, plasmaTTR presented a characteristic band pattern, composed of monomer, anoxidized monomer and several lower isoelectric point (pi) bandscorresponding to different forms of tetramers. A total of 12 plasmasamples (5 controls and 7 carriers TTR V30M) were analyzed in 3 IEFgels. For each treatment condition, a minimum of 4 samples fromdifferent donors were processed. The ratio of TTR tetramer over TotalTTR (TTR tetramer+monomer) was calculated for each plasma sample andrepresented in FIG. 2. This ratio is normally higher for plasma fromnormal individuals than for the plasma from heterozygotic TTR V30Mcarriers plasma, as observed in FIG. 2. Treatment with tolcaponeincreases the amount of TTR tetramer over the monomeric forms comparedto the non treated control plasmas of both normal or mutant TTR; and toa higher extent than tafamidis.

The increase of the tetramer/total TTR ratio induced by the treatmentwith test compounds was pooled for all samples and represented in Table4 as % of stabilization. These values were calculated after normalizingthe tetramer/total TTR ratio obtained for each sample, with the ratioobtained for the non-treated plasma of the corresponding individualdonor as described below:

% stabilization=100×((ratio sample−ratio nt)/ratio nt). Where “ratiosample” is tetramer/total TTR ratio in the presence of compound; and“ratio nt” is tetramer/total TTR ratio of non-treated plasma from samedonor.

TABLE 4 Stability of TTR tetramer in the presence of compounds %stabilization (average +/− sem) Tolcapone 29.9 +/− 7.64 Tafamidis 16.4+/− 5.49 EGCG 51.26 +/− 14.21

Treatment with a TTR stabilizer such as tafamidis or tolcapone increasesthe ratio of tetramer over the monomeric forms. The results shown aboveclearly demonstrate that tolcapone presents a better stabilizationeffect on TTR tetramers than tafamidis.

Example 5: Cell Toxicity Assays

To evaluate TTR-induced cytotoxicity and the preventive effect of thetested compounds, Rat Schwannoma cells (RN22, obtained from AmericanType Cell Collection ATCC), 80% confluent cells in Dulbecco's minimalessential medium with 10% fetal bovine serum, were exposed for 24 hoursto 2 μM of TTR Y78F oligomers. These oligomers were obtained byincubation of soluble TTR Y78F either in the absence or presence of a10× molar excess (final concentration is 20 μM) of test compounds orcontrol (EGCG) at 37° C. for 6 days. Then, cells were trypsinized andcell lysates were used for determination of caspase-3 activation withthe CaspACE fluorimetric 96-well plate assay system (Sigma). Proteinconcentration in lysates was determined with the Bio-Rad protein assaykit.

The results obtained for caspase 3 activity and protein quantificationin each cell culture well are represented in FIG. 3. Extracellularaddition of non-treated TTR Y78F oligomers (control, 01) increasedintracellular levels of Caspase-3, and thus cell death. TTR Y78Foligomers obtained in the presence of compounds that inhibit theformation of toxic oligomeric species (O2-O4) caused lower levels ofCaspase-3 activation in RN22 cells. The reduction of cell toxicity inthe presence of compounds (expressed as 100-% relative to control O1) isshown in table 5. It can be observed that tolcapone showed a greaterreduction of cell cytotoxicity (29%) as compared to tafamidis (12%).

TABLE 5 Reduction of cell toxicity in the presence of compoundsTolcapone 29% Tafamidis 12% EGCG 50%

Example 6: Fibril Disruption

To study the effect of the test compounds on TTR fibrils disruption, weused TTR pre-formed fibrils prepared by incubation of a filtered (0.2 μmfilters) solution of TTR L55P (2 mg/ml in PBS 3.6 μM) for 15 days at 37°C. Subsequently, the samples were incubated either in the absence(control) or presence of a 10× molar excess (36 μM) (finalconcentration) of the test compounds for 4 days at 37° C. The disruptioneffect was evaluated by Transmission Electron Microscopy (TEM) andDynamic Light Scattering (DLS) as previously described (Ferreira et al,2009, FEBS Lett, vol. 583, p. 3569-76).

It was observed that the control sample of TTR pre-formed fibrils(control) is mainly composed by big aggregates and fibrils (particleswith a diameter higher than 1000 nm) and just a small amount of theprotein is in soluble form (particles of 10 nm diameter). As the fibrilsare being disrupted by the tested compounds the relative amount of bigaggregates decrease and the small aggregates and soluble proteinincrease (see FIG. 4).

The fibril disruption activity was quantified from the DLS analysis asthe relative intensity (%) of aggregates and soluble particles after 4days treatment with 36 μM of compounds (table 6).

TABLE 6 DLS Analysis of TTR fibrils relative intensity (%) Solubleparticles Aggregates Aggregates (~10 nm) (~10-100 nm) (~1000 nm) Control28.2 — 71.8 tocalpone 56.1 5.9 38 Tafamidis 35.2 6.7 58.1 EGCG 49.1 26.324.6

It can be observed that samples treated with tolcapone resulted in ahigher amount of small aggregates and soluble proteins, thus exhibitingan important disruption activity. The results also show that tolcaponehas a higher fibril disruption activity than tafamidis.

The results obtained by experiments 1-6 clearly demonstrate thattolcapone has a high inhibitory activity of the formation of TTR amyloidfibrils and such inhibitory activity is higher than tafamidis, which hasbeen described for the treatment of FAP. Further, tolcapone can disruptpre-formed TTR amyloid fibrils more effectively than tafamidis.Altogether, the results indicate that tolcapone can be effectively usedas a a medicament for the treatment of all types of TTR-associatedamyloidosis.

1.-13. (canceled)
 14. A method for disrupting transthyretin (TTR)amyloid fibrils comprising administering to a subject in need thereof acatechol-O-methyltransferase (COMT) inhibitor of formula I or apharmaceutically acceptable salt thereof;

wherein R is selected from the group consisting of —C(O)-PhCH₃,—CH═C(CN)—C(O)—NEt₂ and —CH═C(C(O)CH₃)₂.
 15. The method of claim 14,wherein the COMT inhibitor is tolcapone or a pharmaceutically acceptablesalt thereof.
 16. The method of claim 14, wherein the disruption of TTRamyloid fibrils comprises increasing the amount of soluble protein(particles around 10 nm diameter) and decreasing the amount ofaggregates fibrils with diameters of about 1,000 nm or higher.
 17. Themethod of claim 14, wherein the COMT inhibitor further inhibitsformation of TTR amyloid fibrils.
 18. The method of claim 14, whereinthe COMT inhibitor is administered as an injectable dosage form, as anoral dosage form or as a controlled release dosage form.
 19. The methodof claim 14, wherein an additional therapeutic agent is administered incombination with the catechol-O-methyltransferase (COMT) inhibitor, andwherein the additional therapeutic agent is selected from the groupconsisting of another COMT inhibitor as defined in claim 1, abenzoxazole derivative, iododiflunisal, diflunisal, resveratrol,tauroursodeoxycholic acid, doxocycline and epigallocatechin-3-gallate,wherein the benzoxazole derivative is a compound of formula V

or a pharmaceutically acceptable salt thereof, wherein: Y is selectedfrom the group consisting of COOR, tetrazolyl, CONHOR, B(OH)₂ and OR; Xis O; and R¹, R² and R³ are each independently selected from the groupconsisting of hydrogen, halo, OR, B(OH)₂ and CF₃, and wherein R isselected from the group consisting of hydrogen, C₁-C₆ alkyl, C₁-C₆alkenyl, C₁-C₆ alkynyl, C₁-C₆ cycloalkyl, C₁-C₆ heterocyclyl, phenyl,xylyl, naphthyl, thienyl, indolyl and pyridyl.
 20. The method of claim19, wherein the benzoxazole derivative is a compound of formula VI:

or a pharmaceutically acceptable salt thereof, wherein: Y is selectedfrom the group consisting of COOH, and OH; and R¹, R² and R³ are eachindependently selected from the group consisting of hydrogen, halo, OH,B(OH)₂ and CF₃.
 21. The method of claim 19, wherein the benzoxazolederivative is tafamidis.
 22. The method of claim 19, wherein the atleast one COMT inhibitor and additional therapeutic agent areadministered simultaneously or sequentially.
 23. The method of claim 19,wherein the at least one COMT inhibitor and additional therapeutic agentare administered in two different compositions.
 24. The method of claim14, wherein the COMT inhibitor is administered in an oral dosage form.23. The method of claim 14, wherein the oral dosage form is selectedfrom the group consisting of a tablet, pill, powder, capsule, sachet,liquid syrup, suspension and elixir.
 24. The method of claim 14, whereinbetween about 1 mg/day and about 2,000 mg/day of the at least one COMTinhibitor is administered.